Mechanisms of Epiboly of ectoderm in the Xenopus Laevis embryo Introduction Epiboly is a movement of gastrulation in the amphibian embryo, whereby ectoderm al precursors expand to cover the entire embryo. This process occurs in the surface and deep layer cells in the animal and marginal regions. Three rounds of cell division occur in the deep cells, while they also rearrange to form fewer layers. Superficial cells elongate by cell division while flattening, which gives them greater surface area and thinner depth. The ectoderm eventually covers the entire embryo, internalizing the endoderm.

This process sets up the correct position for the three germ layers, with the ectoderm on the outside, mesoderm in the middle and endoderm on the inside. Keller (1980) found that superficial cells spread, divide, and undergo rearrangements and a temporary change in shape, which produces an increase in area. The deep cells become thinner and decrease in the number of layers. They do this by a process called radial.

Radial is when the deep cells elongate, extend protrusions between one another along radii of the embryo and interdigitate to form fewer layers with greater area. Once this process is complete, the deep region consists of one layer of columnar cells, which flatten and spread to further increase area. In the dorsal marginal zone the cells also undergo a shape change, which is not seen in the cells of the animal region. The difference may be due to the uniform spreading in the animal region contrasted with extension and convergence that occurs in the dorsal marginal zone. In his work on time-lapse films of, Keller (1980) found that the ectoderm becomes corrugated by rapid constrictions of the ap ices of superficial cells and by the appearance of holes in the epithelium.

From this, he suggested that shrinkage, rather than expansion aids in epiboly of the ectoderm. He presents a model (see Figure 1) in which the superficial layer is under tension and the force for expansion must come from the deep cells. The expansion of the deep region is resisted by the tension in the superficial layer resulting in an outward curling of the bi layer (deep and superficial layers). An alternative model (see Figure 2) is also proposed, in which the superficial epithelium is stretched by tension at the margin of the blastopore, which initiates the superficial cells to spread passively (Keller, 1980). While the deep cells rearrange themselves to occupy the areas now available that were once occupied by the superficial cells. Keller (1980) found that the total cell volume and extracellular spaces in these regions remain fairly constant during gastrulation.

An inverse relationship should exist between the thickness and amount of area expansion in a given region. Since the deep region goes through a greater amount of thinning than the superficial layer, it is implied that the spreading of the deep region should also be greater than that of the superficial. From the literature review, there are three alternative hypotheses. The major mechanism of epiboly of the ectoderm may be an increase in cell number produced by an increase in cell division during gastrulation. A different theory proposes that the process occurs mainly by an increase in cell size. Another explanation would be that the ectoderm al spreading is actually occurring by changes in the cell shape.

This three hypotheses could also be combined together, showing that epiboly occurs as a result of all three mechanisms. Methods Embryos of the Xenopus Laevis were collected by normal mating procedures in a Valparaiso University Developmental Biology Lab. Three embryos at stage 10 and three at stage 15 were set in L-Cysteine solution for 3-10 minutes, then placed in Full Strength Steinberg's solution. The embryos were de jellied and devitalized in Full Strength Steinberg's.

At this point, the surface area of the entire embryo was determined by using a micrometer to measure the diameter. The embryos were then placed in a fixative to transport to an electron microscopist. All 6 embryos were graphed with an electron microscope focusing on the surface cells of the animal region. Once graphed, a square region of the cells was used to estimate the surface area. Surface area of the cells was determined by using a ruler to measure the diameter of the cells. An error of approx.

5% was accounted for, since this was a flat surface picture of a curved organism. The mean surface area of the entire embryo was calculated. It was determined from a fate map by Keller (1975) that about 60% of the embryo contains presumptive ectoderm. Therefore, the percentage of the surface area expected to become ectoderm was also calculated. The observed percentage of ectoderm was compared to the expected using a c-square test.

The mean area of the cells was calculated and compared across the two stages. Bibliography Keller, R. E. (1975). Vital Dye Mapping of the Gastrula and Neurula of Xenopus levis: Prospective Areas and Morphogenetic Movements of the Superficial Layer.

Developmental Biology, 42: 222-241. Keller, R. E. (1980). The cellular basis of epiboly: An SEM study of deep-cell rearrange- men during gastrulation in the Xenopus levis. J.

Embryol. exp. Morph. , 60: 201-234. Possible Results and Conclusions One possibility is that the mean cell surface area remains constant across stage 10 and 15. This would mean that the cell size is not increasing during gastrulation and epiboly must be occurring by some other mechanism.

However, if there is a significant change across the stages, then it could be concluded that an increase in cell size contributes to epiboly of the ectoderm. Another possibility is that the mean number of cells may increase across stage 10 and 15. If this is the case, then it could be concluded that an increase in cell number through cell division contributes to the expansion of ectoderm during epiboly. If neither an increase in cell surface area or number is seen, one may conclude that it is a change in cell shape. This result may substantiate Keller's hypothesis that epiboly of the ectoderm is occurring by a shrinkage of the cell, which rearranges (producing a different shape), rather than expansion. A shape change may be seen along with an increase in the number of cells..